Acceptor photobleaching of the three M₁ mAChR cameleons showing primarily plasma membrane localization.

<p>HEK293 cells were transiently transfected with M₁-cam1, M₁-cam2 or M₁-cam5 and imaged by confocal microscopy (458 nm excitation, 470–500 nm emission). Areas of the plasma membrane (delineated by red lines in each image) were bleached using repeated brief exposures to high intensity 514 nm illumination. The graphs show the signal at 458 nm excitation for 470–500 nm emission (ECFPc, cyan line) and >530 nm (EYFP^F46L emission, yellow line), with acceptor photobleach initiated at the arrow. The fluorescence signals from a non-photobleached region were also assessed as a control, which was comparable for all constructs, but only shown for M₁-cam1 (area outlined in cyan within the image and fluorescence within the graph for ECFPc (dark blue) and EYFP^46L (red)). These findings are representative of photobleaching experiments from at least 3 separate transfections for each construct.</p

Cellular localization of M₁-cameleons transiently expressed in HEK293 cells.

<p>HEK293 cells were transiently transfected with M₁-cam1 (<b>A</b>), M₁-cam2 (<b>B</b>), M₁-cam3 (<b>C</b>), M₁-cam4 (<b>D</b>) or M₁-cam5 (<b>E</b>). Images were acquired by confocal microscopy and show fluorescence emission at >530 nm following excitation at 514 nm. Scale bar, 15 µm.</p

Apparent binding affinities (expressed as −log <i>K</i>_B values) for various mAChR agonists and antagonists at the M₁-cam5 AChR, determined by [³H]NMS competition binding.

<p>Data are shown as means ± s.e.m. for duplicate determinations in the number of separate experiments (n) indicated.</p

Comparisons of maximal FRET changes and rate constants for a variety of orthosteric and allosteric ligands in HEK293-M₁-cam5 cells.

<p>Cells were stimulated with a maximally effective concentration of each agonist and FRET changes (<b>A</b>) and K<i>_obs</i> values (<b>B</b>) were determined as described above. Data are presented as means ± s.e.m. from at least three independent experiments. One-way AVOVA (*<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0001).</p

Internalization characteristics of the M₁-cam5 mAChR stably expressed in HEK293 cells.

<p>HEK293-M₁-cam5 cells were treated with various concentration of MCh for 45 min (to assess the concentration-dependency of receptor internalization), or with MCh (300 µM) for 0–60 min (to assess the time-dependency of receptor internalization). Cellular distributions of M₁-cam5 mAChR were monitored by confocal microscopy. For quantification of intracellular fluorescence at least 10 individual cells in five random fields of view were examined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank">Methods</a> section. Data represent means ± s.e.m. from three independent experiments.</p

Overview of M₁ AChR chimeric constructs.

<p>Primers used for introducing <i>AgeI</i> site into the i3 loop are shown where applicable.</p

MCh-induced changes in FRET in HEK293 M₁-cam5 cells.

<p>HEK293 cells stably expressing M₁-cam5 were observed using fluorescence imaging with single wavelength excitation (436 nm) and dual wavelength emission (436 nm to detect ECFPc and 535 nm to detect EYFP^46L. <b>A</b>. Representative images showing plasma membrane distribution of ECFPc (480 nm, left panel) and EYFP^46L (535 nm, middle panel), which overlap (right panel) as expected for signals from the same population of receptors. <b>B–D</b>, right panels: blue and yellow traces represent signals from ECFP and EYFP, respectively; left panels: red traces represent the FRET signal (ratio of F_EYFP/F_ECFP). Addition of MCh (100 µM) induced decreases in FRET, which remained constant throughout the application period (30–40 s; <b>B</b>); this effect was reversed on addition of atropine (1 µM; <b>C</b>); and the MCh-induced change in FRET ratio could be completely prevented by pre-addition of atropine (<b>D</b>). FRET data have been normalized so that the initial FRET signal is 100%. Emission traces are expressed as the change in fluorescence intensity from the basal fluorescence level (<i>F</i>/<i>F</i>₀). Representative traces of at least three independent experiments are shown.</p

Concentration-dependency and dynamics of MCh-induced FRET changes in HEK293-M₁-cam5 cells.

<p><b>A</b>. HEK293 cells stably expressing M₁-cam5 were stimulated with various concentration of MCh (0.3–300 µM) as above, and change in FRET ratio recorded. Data represent the means ± SEM from at least three independent experiments. <b>B</b>. Correlation between the rate constant (K<i>_obs</i>) and MCh concentration was analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank">Methods</a> section. K<i>_obs</i> values were obtained by fitting the FRET data to a single-phase exponential decay. Data represent the means ± s.e.m. from at least three independent experiments.</p

Signal transduction characteristics of M₁-cam5 mAChR.

<p><b>A</b>. Concentration-dependent [³H]IP_x accumulation in wild-type HEK293 (□) or HEK293-M1-cam5 (•) cells stimulated by oxo-M in the presence of 10 mM LiCl. <b>B</b>. Changes in phosphoinositide turnover (phosphatidylinositol 4,5-bisphosphate hydrolysis/Ins(1,4,5)P₃ accumulation) in response to MCh in single HEK293-M₁-cam5 cells using confocal fluorescence imaging. HEK293-M₁-cam5 cells were transfected with the fluorescent biosensor eGFP-PH (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank"><i>Methods</i></a> section). Phosphoinositide turnover was assessed by monitoring the translocation of the eGFP-PH probe from the plasma membrane to the cytoplasm on addition of MCh (10 µM) for 60 s, followed by washout. The trace (B, lower panel) shows a representative time-course of change in cytoplasmic fluorescence intensity for 3–5 cells analyzed per coverslip over three separate experiments. <b>C</b>. MCh-induced activation of the ERK1/2 signalling cascade in HEK293-M₁-cam5 cells. Cells were serum-starved for 24 h and then treated with MCh for 5 min in absence or presence of the mAChR antagonist atropine. A representative western blot is shown for phospho-ERK1/2 and GAPDH (loading control run in parallel) that was repeated independently two more times with similar results.</p

FRET changes induced by mAChR orthosteric and allosteric agonists in HEK293-M₁-cam5 cells.

<p>Addition of arecoline (<b>A</b>; 300 µM), pilocarpine (<b>B</b>; 300 µM), 77-LH-28-1 (<b>C</b>; 10 µM) and AC-42 (<b>D</b>; 30 µM) induced decreases in FRET, which remained constant until reversal by addition of atropine (1 µM). FRET data have been normalized so that the initial FRET signal is 100%.</p